IL-12 Protects against Coxsackievirus B3-Induced Myocarditis by Increasing IFN- γ and Macrophage and Neutrophil Populations in the Heart This information is current as of September 24, 2021. DeLisa Fairweather, Sylvia Frisancho-Kiss, Susy A. Yusung, Masheka A. Barrett, Sarah E. Davis, Ronelle A. Steele, Shannon J. L. Gatewood and Noel R. Rose J Immunol 2005; 174:261-269; ; doi: 10.4049/jimmunol.174.1.261 Downloaded from http://www.jimmunol.org/content/174/1/261 References This article cites 67 articles, 31 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/174/1/261.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 24, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology IL-12 Protects against Coxsackievirus B3-Induced Myocarditis by Increasing IFN-␥ and Macrophage and Neutrophil Populations in the Heart1 DeLisa Fairweather,* Sylvia Frisancho-Kiss,* Susy A. Yusung,* Masheka A. Barrett,* Sarah E. Davis,* Ronelle A. Steele,* Shannon J. L. Gatewood,* and Noel R. Rose2*† Th1-type immune responses, mediated by IL-12-induced IFN-␥, are believed to exacerbate certain autoimmune diseases. We recently found that signaling via IL-12R␤1 increases coxsackievirus B3 (CVB3)-induced myocarditis. In this study, we examined the role of IL-12 on the development of CVB3-induced myocarditis using mice deficient in IL-12p35 that lack IL-12p70. We found that IL-12 deficiency did not prevent myocarditis, but viral replication was significantly increased. Although there were no changes in the total percentage of inflammatory cells in IL-12-deficient hearts compared with wild-type BALB/c controls by FACS analysis, Downloaded from macrophage and neutrophil populations were decreased. This decrease corresponded to reduced TNF-␣ and IFN-␥ levels in the heart, suggesting that macrophage and/or neutrophil populations may be a primary source of TNF-␣ and IFN-␥ during acute CVB3 myocarditis. Increased viral replication in IL-12-deficient mice was not mediated by reduced TNFRp55 signaling, because viral replication was unaltered in TNFRp55-deficient mice. However, STAT4 or IFN-␥ deficiency resulted in significantly in- creased viral replication and significantly reduced TNF-␣ and IFN-␥ levels in the heart, similar to IL-12 deficiency, indicating that http://www.jimmunol.org/ the IL-12/STAT4 pathway of IFN-␥ production is important in limiting CVB3 replication. Furthermore, STAT4 or IFN-␥ defi- ciency also increased chronic CVB3 myocarditis, indicating that therapeutic strategies aimed at reducing Th1-mediated autoim- mune diseases may exacerbate common viral infections such as CVB3 and increase chronic inflammatory heart disease. The Journal of Immunology, 2005, 174: 261–269. iral myocarditis is a frequent cause of sudden death in ␥-mediated mechanisms (6). These findings suggest that IL-12- young adults and can progress to chronic dilated cardio- mediated Th1 responses increase myocarditis. This study further V myopathy (DCM),3 a major cause of heart failure asso- examines the role of IL-12 on the development of CVB3-induced ciated with an autoimmune response (1, 2). Although most people myocarditis. by guest on September 24, 2021 recover from acute viral myocarditis, susceptible individuals de- IL-12 is potent at inducing IFN-␥ production from NK and T velop chronic myocarditis and DCM. Coxsackievirus B3 (CVB3) cells and promotes the differentiation of T cells to a Th1 phenotype infection of susceptible mice results in a disease pattern similar to (7). Th1-mediated immune responses have been implicated in a that observed in humans. Acute myocarditis occurs from days 7 to number of autoimmune diseases, including some forms of colitis, 14 postinfection (p.i.) in all strains of mice, while susceptible mice type I diabetes, multiple sclerosis, rheumatoid arthritis, and myo- (100% prevalence) progress to a chronic phase of disease from day carditis (6, 8, 9). IL-12 is a heterodimer composed of IL-12p35 and 28 to at least 58 p.i. that is associated with the development of IL-12p40 subunits and is secreted as a biologically active IL- DCM (3). Susceptible BALB/c mice respond to CVB3 infection 12p70 molecule primarily by macrophages, neutrophils, and den- with elevated levels of TNF-␣, IL-1␤, and IL-12p70 in the heart dritic cells. Signaling by IL-12 requires coexpression of the IL- during the innate response (4). We have shown that increased ␤ ␤ IL-1␤ and IL-18 levels in the heart correlate with increased acute 12R 1 and IL-12R 2 chains for high affinity IL-12p70 binding ␥ CVB3 myocarditis, and are mediated by TLR4 and IL-12R␤1 sig- and maximal IFN- production. In the mouse, IL-12R signaling naling (5). Recently, CD4ϩ Th1 cells were shown to promote activates STAT1, 3, 4, and 5, with STAT4 believed to be respon- CVB3-induced myocarditis in susceptible BALB/c mice by IFN- sible for most of the biological activities of IL-12p70 through the production of IFN-␥ (7, 10, 11). Similar to IL-12, IL-23 activates STAT1, 3, and 4; induces IFN-␥ production; and exacerbates au- *Department of Pathology and †W. Harry Feinstone Department of Molecular Mi- crobiology and Immunology, Johns Hopkins Medical Institutions, Baltimore, MD toimmune disease (12–15). However, the role of IL-12 and IL-23 21205 is functionally distinct, with IL-12 preferentially affecting naive T Received for publication July 8, 2004. Accepted for publication October 22, 2004. cells, while IL-23 induces proliferation of memory T cells (7). The costs of publication of this article were defrayed in part by the payment of page IL-12 is also required for resistance to certain bacterial, viral, charges. This article must therefore be hereby marked advertisement in accordance and intracellular parasitic infections (16–19). IL-12 exerts its in- with 18 U.S.C. Section 1734 solely to indicate this fact. fluence over many infectious agents by stimulating the production 1 This work was supported by National Institutes of Health Grants HL67290, HL70729, and AI51835. of TNF-␣ and IFN-␥, which act synergistically to control infec- 2 Address correspondence and reprint requests to Dr. Noel R. Rose, Department of tions by recruiting and activating macrophages, NK cells, and T Pathology, Johns Hopkins Medical Institutions, Ross Building, Room 659, 720 Rut- cells to perform their effector functions (17, 20–23). IL-12-in- land Avenue, Baltimore, MD 21205. E-mail address: nrrose@jhmi.edu duced IFN-␥ appears to act on different effector cells in different 3 Abbreviations used in this paper: DCM, dilated cardiomyopathy; CVB3, coxsack- ievirus B3; EAM, experimental autoimmune myocarditis; LCMV, lymphocytic cho- viral infections. For example, CD8 CTLs have been shown to re- riomeningitis virus; p.i., postinfection. duce lymphocytic choriomeningitis virus (LCMV) infection (18), Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 262 IL-12 REDUCES MYOCARDITIS while NK cells play a major role in defense against murine cyto- Cytokine measurement megalovirus infection (3, 24). Although clearance of viral infec- Hearts were frozen in dry ice immediately and stored at Ϫ80°C until ho- tions is usually thought to be mediated by destruction of infected mogenized. Tissues were homogenized at 10% weight/volume in 2% cells by effector immune cells, it is now becoming clear that re- MEM, and supernatants were stored at Ϫ80°C until used in ELISAs or lease of IFNs and TNF at the site of infection is also important in plaque assays. Cytokines were measured in heart supernatants using Quan- purging virus from infected cells noncytopathically (22, 25, 26). tikine cytokine ELISA kits purchased from R&D Systems, according to manufacturer’s instructions. The limits of detection for the cytokine kits Cytokines can also control viral infections indirectly by modulat- were as follows: TNF-␣, 5.1 pg/ml; IL-1␤, 3 pg/ml; IL-12, 2.5 pg/ml; ing the immune response and by up-regulating Ag processing and IL-18, 25 pg/ml; and IFN-␥, 2 pg/ml. Cytokines were below detectable display of viral epitopes on the surface of infected cells (22). How- levels in the 2% MEM used to homogenate samples (data not shown). Ϯ ever, the role of IL-12 in the development of CVB3-induced myo- Cytokines were expressed as pg/g heart tissue SEM. carditis has not been previously investigated. Plaque assay In the present study, we examine the role of IL-12 on the de- The level of infectious virus was determined in individual homogenates by velopment of CVB3-induced myocarditis using mice deficient in plaque assay according to standard procedures (5, 28). Samples were pro- IL-12p35, STAT4, or IFN-␥. We found that IL-12 deficiency did cessed in the same manner as for cytokine analysis. Dilutions of tissue not prevent the development of acute myocarditis, but allowed a supernatants were incubated on confluent Vero cell (ATCC) monolayers significant increase in viral replication in the heart. STAT4 or for1hat37°C and 5% CO2 to allow viral attachment, and then incubated ␥ for 3 days to allow plaque formation. Virus titers were expressed as the IFN- deficiency also resulted in significantly increased levels of mean PFU/g tissue Ϯ SEM, and the limit of detection was 10 PFU/g tissue.